1. Field of the Invention
This invention relates to a reporter mechanism useful in analytical chemistry. More particularly, it relates to a reporter mechanism for use in conjunction with specific binding assay techniques to achieve highly sensitive determinations of analytes in test samples.
Specific binding assay techniques are useful for determining various substances of diagnostic, medical, environmental and industrial importance which appear at very low concentrations. Specific binding assays are based on a specific interaction between the analyte under determination and a binding partner therefor. Where one of the analyte and its binding partner is an antibody and the other is a corresponding hapten or antigen, the assay is known as an immunoassay.
In such assays, it is necessary to have a reporter mechanism or event, for example, measurement of a label, which can give a measurable indication of the extent to which the specific binding reaction takes place. For example, radioactive or fluorescent labels can be present on the binding partner and the presence of this radioactive or fluorescent label in the specifically bound pair can be used to detect formation of the pair. Alternatively, the specific binding reaction can give rise to a change in the detectable properties of the reporter group, for example by increasing or decreasing a measurable property.
Reporter mechanisms and labels are employed in heterogeneous analysis settings and homogeneous assay settings. A heterogeneous assay is one in which the labeled specifically bound pair is physically separated from unbound labels. A homogeneous assay is one in which the labeled pairs are not separated from the unbound labels but the bound labels and unbound labels are distinguishable from one another. See J. BIOL. CHEM., 251, 4172-8 (1976).
2. Description of Prior Art
The present reporter mechanism involves interligand transfer of metals. Metals have been used in reporter mechanisms heretofore. In one elementary art-taught method, a species containing a metal ion is determined by measuring the amount of the metal in the sample by conventional wet chemistry quantitative or qualitative analysis methods.
In another method of the art a nonmetal-containing species is determined by reacting it with a metal-containing material in a biospecific reaction to form a metal-containing specific binding pair and thereafter determining the amount of metal associated with the pair and thus indirectly the amount of the nonmetal species. One embodiment of such a process is shown in U.S. Pat. No. 4,341,957, issued Jul. 27, 1982 in which analysis methods involving an antibody labeled with a fluorescent rare earth metal chelate reacting with its corresponding antigen are shown. Thereafter the amount of fluorescent chelate associated with the pair is determined fluorometrically. Some references generally to tagging of biospecific molecules with metal ions include, for example, U.S. Pat. Nos. 4,283,382; 4,352,751; 4,374,120; and 4,565,790, Europoean patent application Nos. 0154788, 0159066, and 064484 as well as Japanese Pat. No. 57,149,965 and German patent application No. 3239410-Al.
U.S. Pat. Nos. 4,058,732 and 4,150,295, issued on Nov. 15, 1977 and Apr. 17, 1979, respectively and a chapter appearing at pages 67-80 of Immunofluorescence and Related Staining Techniques, Knapp, et al., eds. (1978, Elsevier/North Holland Biomedical Press) describe the use of fluorescent background rejection in assay techniques and disclose that rare earth metal chelates are attractive fluorescent species. This art-taught background rejection relies on time gating and measures longlived fluorescence from the assayed species after shortlived background fluorescence has decayed. This background rejection technique is employed in certain embodiments of this invention.
U.S. Pat. No. 4,352,751 issued on Oct. 5, 1979 discloses a family of rare earth metal chelates and their use in fluorescent background rejection techniques. Similarly, European patent application No. 68875 A2 filed on Jun. 28, 1982 of Eastman Kodak Co. discloses fluorescent rare earth chelate labels in fluoroassay techniques.
ANALYTICAL BIOCHEMISTRY, 137, 335 (1984) and European patent application No. 64484 A2 of Wallac Oy disclose a heterogeneous assay method named the "dissociation-enhanced lanthanide fluoroimmunoassay" or "DELFIA". These assays are either solid phase sandwich assays or solid phase competitive assays. In the first case, solid phase first antibody binds a two-site antigen which then binds a europium chelate-labeled second antibody. In the second case, solid phase antigen competes with free antigen for a limited amount of free europium chelate-labeled antibody. In either case, following a separation step, the europium-labeled antibody ends up on the solid phase. The materials used in these methods give a relatively low fluorescence chelate which is not detectable at desired levels of sensitivity. Thereafter the solid phase chelate is treated with an acidic (pH about 3.2) enhancement solution which causes the metal to be released from the solid chelate into solution. The enhancement solution also contains a .beta.-diketone which chelates the freed ions leading to development of fluorescence. Because of insolubility of the .beta.-diketone in aqueous solutions, the enhancement solution also must contain a detergent (Triton X-100 ) to solubilize both the .beta.-diketone and an added synergistic agent (tri-N-octylphosphine oxide, TOPO,) which is also water immiscible. These materials are employed to yield a micellar system and rely upon the release of the metal ion from the first complex and its later chelation by the .beta.-diketone to provide the detectable fluorescent species. The lanthanide ion label that is released from the chelate by an acid treatment can be measured using the fluorescence background rejection technique.
Other references of interest include articles appearing at CLIN. CHEM., Winston Salem, N.C., 29(1) 65-8 (1983) and PROC. NATL. MEET. BIOPHYS. MED. ENG, FINL., 4, 199-202 (1982), European patent application No. 103558 published Mar. 21, 1984, and U.S. Pat. No. 4,374,120 all from the Wallac Oy group; and Kodak's U.S. Pat. No. 4,283,382, issued Aug. 11, 1981.
Other art to be noted includes the article "A Review of Fluoroimmunoassay and Immunofluorometric Assay", D. S. Smith et al, ANN. CLIN. BIOCHEM., 18, (1981) 253-274 which provides a summary of the heterogeneous and homogeneous fluoroassay techniques proposed heretofore; U.S. Pat. Nos. 3,998,943; 4,020,151; 3,939,350; 4,220,450 and 3,901,654 which show analytical assay techniques. A number of additional references to various general homogeneous assay techniques are provided in the patent application filed of even date herewith and incorporated herein by reference.